Natural plant products have served as tremendously valuable tools for deciphering cellular and molecular mechanisms contributing to somatosensation, nociception, and pain. Notable examples include the use of natural analgesics, such as morphine (from the opium poppy) and salicylate (from willow bark) to discover opioid receptors and cyclooxgenases, respectively. Other important examples include the use of natural irritants, such as capsaicin (from chili peppers) and menthol (from mint leaves) to identify ion channels that detect heat and cold, respectively. Indeed, each of these proteins represents a validated or potential target for pharmacological management of acute or chronic pain. Plants are not unique in their capacity to produce chemical agents that target sensory neurons or other excitable cells. Indeed, venoms from vertebrate organisms (ranging from crustaceans to mammals) represent a vast pharmacopoeia that has great potential to yield novel agents with which to identify or characterize receptors, ion channels, or other signaling molecules that contribute to sensory transduction. With this in mind, we have recently discovered a novel family of peptide toxins from tarantula that activate the capsaicin receptor, TRPV1, to produce inflammatory pain (presumably as part of the spider's defensive strategy to ward off predators). These so-called vanillotoxins are the first peptide toxins known to interact with TRPV1, or for that matter, any member of the extended family of excitatory TRP channels. This proposal builds on our initial discovery of the vanillotoxins, with the goal of using natural toxins and synthetic derivatives to probe the structure and/or function of TRP channels or other receptors expressed by primary afferent neurons or other types of excitable cells. The first aim of this proposal is geared towards exploiting the vanillotoxins as novel biochemical tools with which to probe TRPV1 channel structure, beginning with experiments designed to map sites of vanillotoxin-TRPV1 interaction. Little is currently known about TRP channel architecture or gating mechanisms, and comparison of our findings with data from the voltage-gated channel field will provide valuable insights into these and related structure-function questions. The second and third aims are directed towards identifying and characterizing novel toxins from a variety of venomous creatures that target TRPV1 or other sites on excitable cells. This will expand the repertoire of pharmacological agents with which to study known or novel sensory receptors, including those that contribute to the very important, but still enigmatic process of mechanotransduction. PUBLIC HEALTH RELEVANCE Cell surface receptors and ion channels play essential roles in cellular communication throughout the body, including many aspects of nervous system and cardiovascular function. In this proposal, we describe strategies for exploiting natural toxins as new biochemical tools with which to study the structure and function of receptors and ion channels that contribute to the detection of sensory stimuli, particularly in relationship to pain sensation. Results from this work will provide a more in-depth understanding of how these receptors and channels respond to noxious (pain-producing) stimuli under normal and pathological conditions, all of which is relevant to elucidating processes that contribute to acute and chronic pain. Results from these studies may provide insights into strategies for developing novel therapeutic agents or protocols for the treatment of chronic pain syndromes.